lambda protein phosphatase kit  (New England Biolabs)

Journal: bioRxiv

Article Title: Two orthogonal MAP3K-driven pathways of NLRP1 inflammasome activation revealed by poisonous beetles

doi: 10.64898/2026.01.23.701189

Figure Lengend Snippet: TAK1 directly phosphorylates the NLRP1 DR at specific sites and requires a specific TST motif for NLRP1 inflammasome activation. A. Rhodamine labelled recombinant SNAP-tagged NLRP1-DR following PhosTag-SDS-Agarose-PAGE. Recombinant NLRP1-DR was incubated with recombinant ZAKα, p38α, and TAK1-TAB1 fusion in a standard kinase reaction for 60 minutes and labelled with SNAP ligand fluorescence (TMR-STAR). Lambda protein phosphatase or a negative control was spiked into each reaction, as indicated. Phosphorylated vs unphosphorylated species are indicated. B. IL-1β ELISA of NLRP1 KO N/TERTs overexpressing GFP_NLRP1-86-C_FLAG (WT), SATA 1-4, or STless mutants following treatment with 50 μM cantharidin or 1 μM anisomycin. Cell culture media was harvested after 8 hours of treatment. C. Recombinant SNAP-tagged NLRP1-DR was incubated with recombinant TAK1-TAB1 fusion or ZAKα in a standard kinase reaction for 60 minutes and mass spectrometry was used to detect phosphorylated peptides. Phosphorylated residues detected by mass spectrometry are indicated with solid dots while residues identified from the following add-back analysis are indicated with clear dots. Residues phosphorylated by TAK1 and ZAKα are indicated with blue and red respectively. D. Rhodamine labelled SNAP-tagged NLRP1-DR ‘Add-back’ mutants following PhosTag-SDS-Agarose-PAGE. In the STless background, indicated alanine residues were mutated back to serine or threonine. These recombinantly expressed mutants were incubated with recombinant TAK1-TAB1 fusion or ZAKα in a standard in vitro kinase reaction for 60 minutes. Phosphorylated vs unphosphorylated species for each protein is indicated. The crucial “TZ2” motif is highlighted in red. E. Immunoblot of cleaved PARP1, GSDMD (full length and cleaved), GSDME (full length and cleaved), GAPDH, Pro-IL-1β, cleaved IL-1β, and IL-18 (full length and cleaved) or F. IL-18 and IL-1β ELISA from NLRP1 KO N/TERTs overexpressing GFP_NLRP1-86-C_FLAG (WT), TZ2 mutant, or GFP control following treatment with 1 μM anisomycin, DMSO (vehicle control), and 50 μM cantharidin. Cells, floaters and cell culture media were harvested after 8 hours of treatment. Error bars represent SEM from three technical replicates, where one replicate refers to an independent sample. Significance values were calculated based on one-way ANOVA followed by Dunnett’s test for multiple pairwise comparisons (B) or two-way ANOVA followed by Sidak’s test for multiple pairwise comparisons (F). ns, nonsignificant; *P < 0.05; ****P < 0.0001.

Article Snippet: For lambda phosphatase treatments, a final concentration of 1X NEBuffer for Protein MetalloPhosphatases (PMP), 1 mM MnCl 2 , and 0.4 μL of Lambda Protein Phosphatase (NEB, P0753S) was added to the reaction mix and incubated at 30 °C for 30 minutes.

Techniques: Activation Assay, Recombinant, Incubation, Fluorescence, Negative Control, Enzyme-linked Immunosorbent Assay, Cell Culture, Mass Spectrometry, In Vitro, Western Blot, Mutagenesis, Control